脂联素与非酒精性脂肪肝炎的关系探讨
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摘要
非酒精性脂肪性肝病(nonalcoholic fatty liver disease, NAFLD)是病理改变与酒精性肝病相似但无过量饮酒史的临床病理综合征,其疾病谱包括单纯性脂肪肝、非酒精性脂肪性肝炎(nonalcoholic steatohepatitis, NASH)、脂肪性肝纤维化/肝硬化,甚至并发肝癌,严重威胁人类健康。近年我国NAFLD的发病率呈逐渐上升趋势,NASH作为单纯脂肪肝向非酒精性肝纤维化发展的必经阶段,其发病机制尚未完全清楚,因此亦缺乏有效的防治策略。脂联素由脂肪细胞分泌,是目前发现的唯一与血脂水平呈负相关的胶原样细胞因子,具有调节脂代谢、调节糖代谢、抗炎等作用。脂联素有两种受体:AdipoR1和AdipoR2,其中AdipoR2在肝脏中表达丰富,提示脂联素可能参与NASH发生发展过程。噻唑烷二酮类药物(罗格列酮)为特异性PPARγ激动剂,我们的前期研究发现其具有改善蛋氨酸-胆碱缺乏(methionine-choline deficient diet, MCD)饮食诱导的小鼠肝脂肪变、炎症及肝纤维化,但脂联素在此模型中的变化及其在NASH进展中的作用及机制尚不清楚。本研究采用MCD饮食建立小鼠NASH模型,以罗格列酮进行干预实验,探讨脂联素在NASH发病及进展中的作用及作用机制,为NAFLD的靶向性药物/基因治疗提供新的途径。
目的:采用蛋氨酸-胆碱缺乏(MCD)饮食建立小鼠NASH模型,应用罗格列酮进行干预治疗,观察脂联素、TNF-α、IL-6的表达变化与疾病进展的关系,以明确脂联素在NASH发病中的作用及作用机制,以及PPARγ激动剂调节脂联素表达、阻滞NASH进展的作用,为临床选择靶向性治疗非酒精性脂肪性肝炎的药物或基因治疗提供理论依据。
方法:选用8周龄健康雄性C57BL6/J小鼠30只,随机分为3组,每组10只。模型组采用MCD饲料,对照组给予胆碱、蛋氨酸充足的饲料,罗格列酮干预组应用MCD饲料加罗格列酮(50mg/kg/d),共喂养3周。血清丙氨酸氨基转移酶(alanine aminotransferase, ALT),甘油三酯(triglyceride, TG)采用日本Olympus AU 2700全自动生化分析仪测定;采用苏木精-伊红(hematoxylin-eosin, HE)染色及Masson染色观察肝组织病理学改变;肝组织脂联素表达采用免疫组织化学染色方法检测;TNF-α、IL-6 mRNA和蛋白表达分别采用RT-PCR和Western blot检测。
结果:
1动物一般情况:对照组小鼠活泼、毛发有光泽,体重逐渐增加。模型组和罗格列酮干预组小鼠体重均下降,尤以模型组明显。对照组、模型组和干预组小鼠体重、肝湿重分别为:26.00±1.00 g、14.14±0.68 g、14.31±0.81 g,0.89±0.12 g、0.53±0.10 g、0.56±0.10 g。模型组、罗格列酮干预组小鼠体重、肝湿重较对照组明显下降(P值均<0.05),而罗格列酮干预组与模型组相比无统计学差异(P>0.05)。对照组、模型组和干预组小鼠肝脏指数分别为:0.034±0.005、0.038±0.006、0.039±0.008,各组相比无统计学差异(P>0.05)。
2血清生化学改变:对照组、模型组和干预组小鼠血清ALT、TG水平依次为:50.78±10.03 U/L、161.56±28.23 U/L、83.56±12.27 U/L;0.54±0.29 mmol/L、0.97±0.81 mmol/L、0.76±0.13 mmol/L。模型组小鼠血清ALT、TG均显著高于对照组(P<0.01),而罗格列酮干预后可明显降低ALT、TG水平(P<0.01)。
3肝组织病理学变化:肉眼观察:对照组小鼠肝脏为红褐色,明亮有光泽。模型组小鼠肝脏体积明显减小,整个肝脏呈浅黄色,与周围组织有粘连,切面油腻、无光泽。罗格列酮干预组小鼠肝脏体积亦有缩小,为红褐色、有光泽。光镜观察:对照组小鼠肝脏肝小叶结构正常,肝板排列整齐;模型组肝细胞弥漫性水样变,呈现大泡性为主的肝细胞脂肪变,以腺泡3带明显,小叶内可见点状或灶状肝细胞坏死伴以单个核细胞为主的混合性炎细胞浸润,可见少量分叶核白细胞。汇管区及窦周纤维组织增生不明显(F2~3G2~3S0~1)。罗格列酮干预组小鼠肝损伤较模型组明显减轻,脂肪变肝细胞明显减少,炎症、坏死及纤维化程度明显减轻(F1G1~2S0~1)。
4肝组织TNF-αmRNA的表达:与对照组相比,模型组TNF-αmRNA表达明显增强(0.82±0.17 vs. 1.26±0.33, P <0.05),而罗格列酮干预组表达较模型组减弱(1.26±0.33 vs. 0.94±0.14, P<0.05),差异具有统计学意义。
5肝组织TNF-α蛋白水平表达情况:与对照组相比,模型组TNF-α蛋白表达明显增加(0.47±0.06 vs. 1.21±0.05, P<0.05),而罗格列酮干预组表达较模型组明显减少(1.21±0.05 vs. 0.83±0.03, P<0.05),差异具有统计学意义。
6肝组织IL-6 mRNA表达情况:与对照组相比,模型组IL-6 mRNA表达显著增加(0.34±0.01 vs. 0.89±0.01, P<0.01),而罗格列酮干预组表达较模型组明显下降(0.89±0.01 vs. 0.57±0.03, P<0.05),差异具有统计学意义。
7小鼠肝组织IL-6蛋白表达情况:与对照组相比,模型组IL-6蛋白表达显著增加(0.46±0.01 vs. 0.91±0.01, P <0.01),而罗格列酮干预组表达较模型组明显下降(0.91±0.01 vs. 0.79±0.02, P<0.05),差异具有统计学意义。
8肝组织脂联素的表达变化:对照组小鼠肝组织脂联素在小叶中央静脉周围及窦内皮细胞有少量表达;与对照组相比,模型组肝脏脂联素表达明显增强,且多表达于脂肪变肝细胞胞浆、炎症细胞浸润区枯否细胞胞浆及窦内皮细胞胞浆中,(1.46±0.27 vs. 15.24±0.36, P<0.01);罗格列酮干预组脂联素阳性表达较模型组明显减少(15.24±0.36 vs. 9.46±0.74, P< 0.05),差异具有统计学意义。
结论:
1非酒精性脂肪性肝炎小鼠肝组织中脂联素表达明显上调,并与TNF-α及IL-6表达呈一致趋势,提示在NASH小鼠肝脂肪变及炎症的发生及进展中,脂联素可能通过代偿性表达增加来拮抗炎性因子的表达,从而阻止疾病的进展。
2罗格列酮可上调PPARγ基因的表达,进一步上调脂联素表达水平、抑制炎症因子TNF-α、IL-6的表达,从而延缓或阻止NASH的发生与进展。
Nonalcoholic fatty liver disease (NAFLD) is a clinical and pathological syndrome who is similar with alcoholic liver disease in pathologic histology but in the absence of significant alcohol consumption. The spectrum of it includes simple liver steatosis, nonalcoholic steatohepatitis (NASH) and fibrosis/cirrhosis in order. Even it can progress to hepatocellular carcinoma. So it is considered as a great threat against people’s health. The prevalence of NAFLD is increasing year by year. NASH, as a key process from simple steatosis to fibrosis/cirrhosis, has not been clearly known in its pathogenesis, so an efficient approach has not been found by now. Adiponectin, a gelatin-binding hormone secreted by adipose tissue, is the only cytokine who is negatively correlated with lipid level in blood serum. Adiponectin plays an important role in regulating lipid metabolism and glycometabolism, ameliorating inflammation. Two adiponectin receptors have been found so far: AipoR1 and AdipoR2. AdipoR2 mostly expressis in liver. So it is supposed that adiponectin may participate in the formation and development of NASH. Thiazolidinediones (rosigliazone) is the specific agonist of peroxisome proliferator-activated receptor gamma (PPARγ). Our previous research showed that rosigliazone could inhibite the hepatic steatosis, inflammation and fibrosis/cirrhosis of mice induced with methionine-choline deficient (MCD) diet. But what is the role of adiponectin in the model is not clear. In this study was to investigate effect of adiponectin in MCD feeding mice and rosigliazone treated mice, which may provide a new approach of NASH therapy through gene modulation.
Objective: In this study, experimental nonalcoholic steatohepatitis models were established by feeding male C57BL6/J mice with methionine-choline deficient (MCD) diet. The treatment group mice were administrated with MCD diet combined with rosigliazone. The expression of adiponectin, tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) were detected to clarify role and mechanism of PPARγagonist, rosigliazone in NASH. This study will provide an theoretical basis for target gene theropy on NASH.
Methods: Experimental models of NASH were established by feeding mice with MCD diet. Thirty healthy male C57BL6/J mice were fed with methionine-choline supplemented diet for one week, and then were randomly divided into three groups: control group, were fed with methionine-choline supplemented diet, model group (MCD group), were given MCD diet, treatment group (MCD+R group), used MCD diet combined with rosiglitazone (50mg/kg/d) for 3 weeks. Serum alanine aminotransferase (ALT) and triglyceride (TG) were tested by enzymic method with automatic biochemistry analyzer. The grade of hepatic steatosis, inflammation and fibrosi were observed by Hematoxylin and eosin, and Masson trichromatism stained paraffin-embedded liver tissues isolated from mice. Adiponectin expression in the liver was examined by immunohistochemistry. The expression of TNF-αand IL-6 mRNA and protein were analyzed by RT-PCR. and western blot, respectively.
Results:
1 The common behavior of mice: control mice were active, their hair was bright and weight increased gradually. Body weight of mice in model group and rosiglitazone group decreased remarkably. Body weight and liver weight in control group, model group and treatment group were respectively :26.00±1.00 g 14.14±0.68 g, 14.31±0.81 g,0.89±0.12 g 0.53±0.10 g 0.56±0.10 g. Body weight and liver weight in model group and treatment group were notedly decreased compared with control group (both P value <0.05). The liver indexs of control group, model group and treatment group was respectively 0.034±0.005, 0.038±0.006, 0.039±0.008, there was no difference among the three groups (P >0.05).
2 The test of biochemical markers of serum: ALT level was markedly elevated in MCD diet mice compared with control mice (50.78±10.03 U/L vs. 161.56±28.23 U/L,P<0.01), and a significant reduction was noticed after rosiglitazone treatment (161.56±28.23 U/L vs. 83.56±12.27 U/L, P<0.01). So does the three groups’serum TG content, control: 0.54±0.29 mmol/L, models: 0.97±0.81 mmol/L, treated group: 0.76±0.13 mmol/L, P <0.05.
3 Liver histopathology : macroscopic observation: Livers in control group were in reddish-brown, with bright gloss. Livers in model group were much smaller than in control group. The whole livers were in light yellow and adhered with other tissues. The cut surface looked reddish-brown without gloss. Livers in treat group were little smaller than in control group and looked reddish-brown with gloss. light microscope observation: Histology of the livers were normal in control mice. Hepatic plates looked well. However, severe steatohepatitis was developed in mice fed with MCD diet, the hepatocytes presented severe macrovesicular steatosis and lacated mainly in zone 3 near the central veins. Spotty necrosis and confluent necrosis can be seen in hepatic lobulars, where mixed inflammatory infiltration with lymphocytes and polymorphonuclear may take place. There were not obvious hepatic fibrosis in perisinusoidal and portal areas (F2-3G2-3S0-1). The degree of hepatic steatosis and inflammation is dramatically ameliorated by administrated rosiglitazone in MCD fed mice (F1G1-2S0-1).
4 The expression of TNF-αmRNA was dramatically improved in MCD fed animals compared with the control group (0.82±0.17 vs. 1.26±0.33,P <0.05), whereas which was dramatically decreased in rosiglitazone treated animals (1.26±0.33 vs. 0.94±0.14,P<0.05).
5 The expression of TNF-αprotein was increased in MCD mice compared with control group (0.47±0.06 vs. 1.21±0.05,P<0.05), whereas which was dramatically decreased in rosiglitazone treated mice (1.21±0.05 vs. 0.83±0.03,P<0.05).
6 The expression of IL-6 mRNA was up-regulated in MCD group (0.34±0.01 vs. 0.89±0.01 , P<0.01) and could be down-regulated by rosiglitazone (0.89±0.01 vs. 0.57±0.03,P<0.05).
7 The expression of IL-6 protein was up-regulated in model animals (0.46±0.01 vs. 0.91±0.01 , P<0.01) and could be down-regulated by rosiglitazone in treatment group (0.91±0.01 vs. 0.79±0.02,P<0.05).
8 Hepatic adiponectin expression: Positive expression was presented around central veins and in endochylema of perisinusoidal cells in the livers of control group. But in model group positive expressions are dramatically enhanced and mainly express in the endochylema of steatosis hepatocytes, kupffer cells in inflammatory infiltration areas and perisinusoidal cells. (1.46±0.27 vs. 15.24±0.36,P<0.01). Compared with model group, the expression of adiponectin was decreased by using rosiglitazone. (15.24±0.36 vs. 9.46±0.74,P< 0.05).
Conclusion:
1 The expressions of adiponectin is dramatically enhanced in livers of the MCD fed mice, which was consistent with expressions of TNF-αand IL-6, which indicates that adiponectin through compensated up-regulation might prevent expression of inflammatory factors and inhibit NASH progression.
2 Rosiglitazone could up-regulate PPARγexpression, further up-regulate adiponectin expression, down-regulate expression of TNF-αand IL-6. Thus, rosiglitazone can inhibit hepatic steatosis, inflammation and fibrosis induced by MCD diet by modulating PPARγ, adiponectin and related inflammatory factor expression.
目的:采用蛋氨酸-胆碱缺乏(MCD)饮食建立小鼠NASH模型,应用罗格列酮进行干预治疗,观察脂联素、TNF-α、IL-6的表达变化与疾病进展的关系,以明确脂联素在NASH发病中的作用及作用机制,以及PPARγ激动剂调节脂联素表达、阻滞NASH进展的作用,为临床选择靶向性治疗非酒精性脂肪性肝炎的药物或基因治疗提供理论依据。
方法:选用8周龄健康雄性C57BL6/J小鼠30只,随机分为3组,每组10只。模型组采用MCD饲料,对照组给予胆碱、蛋氨酸充足的饲料,罗格列酮干预组应用MCD饲料加罗格列酮(50mg/kg/d),共喂养3周。血清丙氨酸氨基转移酶(alanine aminotransferase, ALT),甘油三酯(triglyceride, TG)采用日本Olympus AU 2700全自动生化分析仪测定;采用苏木精-伊红(hematoxylin-eosin, HE)染色及Masson染色观察肝组织病理学改变;肝组织脂联素表达采用免疫组织化学染色方法检测;TNF-α、IL-6 mRNA和蛋白表达分别采用RT-PCR和Western blot检测。
结果:
1动物一般情况:对照组小鼠活泼、毛发有光泽,体重逐渐增加。模型组和罗格列酮干预组小鼠体重均下降,尤以模型组明显。对照组、模型组和干预组小鼠体重、肝湿重分别为:26.00±1.00 g、14.14±0.68 g、14.31±0.81 g,0.89±0.12 g、0.53±0.10 g、0.56±0.10 g。模型组、罗格列酮干预组小鼠体重、肝湿重较对照组明显下降(P值均<0.05),而罗格列酮干预组与模型组相比无统计学差异(P>0.05)。对照组、模型组和干预组小鼠肝脏指数分别为:0.034±0.005、0.038±0.006、0.039±0.008,各组相比无统计学差异(P>0.05)。
2血清生化学改变:对照组、模型组和干预组小鼠血清ALT、TG水平依次为:50.78±10.03 U/L、161.56±28.23 U/L、83.56±12.27 U/L;0.54±0.29 mmol/L、0.97±0.81 mmol/L、0.76±0.13 mmol/L。模型组小鼠血清ALT、TG均显著高于对照组(P<0.01),而罗格列酮干预后可明显降低ALT、TG水平(P<0.01)。
3肝组织病理学变化:肉眼观察:对照组小鼠肝脏为红褐色,明亮有光泽。模型组小鼠肝脏体积明显减小,整个肝脏呈浅黄色,与周围组织有粘连,切面油腻、无光泽。罗格列酮干预组小鼠肝脏体积亦有缩小,为红褐色、有光泽。光镜观察:对照组小鼠肝脏肝小叶结构正常,肝板排列整齐;模型组肝细胞弥漫性水样变,呈现大泡性为主的肝细胞脂肪变,以腺泡3带明显,小叶内可见点状或灶状肝细胞坏死伴以单个核细胞为主的混合性炎细胞浸润,可见少量分叶核白细胞。汇管区及窦周纤维组织增生不明显(F2~3G2~3S0~1)。罗格列酮干预组小鼠肝损伤较模型组明显减轻,脂肪变肝细胞明显减少,炎症、坏死及纤维化程度明显减轻(F1G1~2S0~1)。
4肝组织TNF-αmRNA的表达:与对照组相比,模型组TNF-αmRNA表达明显增强(0.82±0.17 vs. 1.26±0.33, P <0.05),而罗格列酮干预组表达较模型组减弱(1.26±0.33 vs. 0.94±0.14, P<0.05),差异具有统计学意义。
5肝组织TNF-α蛋白水平表达情况:与对照组相比,模型组TNF-α蛋白表达明显增加(0.47±0.06 vs. 1.21±0.05, P<0.05),而罗格列酮干预组表达较模型组明显减少(1.21±0.05 vs. 0.83±0.03, P<0.05),差异具有统计学意义。
6肝组织IL-6 mRNA表达情况:与对照组相比,模型组IL-6 mRNA表达显著增加(0.34±0.01 vs. 0.89±0.01, P<0.01),而罗格列酮干预组表达较模型组明显下降(0.89±0.01 vs. 0.57±0.03, P<0.05),差异具有统计学意义。
7小鼠肝组织IL-6蛋白表达情况:与对照组相比,模型组IL-6蛋白表达显著增加(0.46±0.01 vs. 0.91±0.01, P <0.01),而罗格列酮干预组表达较模型组明显下降(0.91±0.01 vs. 0.79±0.02, P<0.05),差异具有统计学意义。
8肝组织脂联素的表达变化:对照组小鼠肝组织脂联素在小叶中央静脉周围及窦内皮细胞有少量表达;与对照组相比,模型组肝脏脂联素表达明显增强,且多表达于脂肪变肝细胞胞浆、炎症细胞浸润区枯否细胞胞浆及窦内皮细胞胞浆中,(1.46±0.27 vs. 15.24±0.36, P<0.01);罗格列酮干预组脂联素阳性表达较模型组明显减少(15.24±0.36 vs. 9.46±0.74, P< 0.05),差异具有统计学意义。
结论:
1非酒精性脂肪性肝炎小鼠肝组织中脂联素表达明显上调,并与TNF-α及IL-6表达呈一致趋势,提示在NASH小鼠肝脂肪变及炎症的发生及进展中,脂联素可能通过代偿性表达增加来拮抗炎性因子的表达,从而阻止疾病的进展。
2罗格列酮可上调PPARγ基因的表达,进一步上调脂联素表达水平、抑制炎症因子TNF-α、IL-6的表达,从而延缓或阻止NASH的发生与进展。
Nonalcoholic fatty liver disease (NAFLD) is a clinical and pathological syndrome who is similar with alcoholic liver disease in pathologic histology but in the absence of significant alcohol consumption. The spectrum of it includes simple liver steatosis, nonalcoholic steatohepatitis (NASH) and fibrosis/cirrhosis in order. Even it can progress to hepatocellular carcinoma. So it is considered as a great threat against people’s health. The prevalence of NAFLD is increasing year by year. NASH, as a key process from simple steatosis to fibrosis/cirrhosis, has not been clearly known in its pathogenesis, so an efficient approach has not been found by now. Adiponectin, a gelatin-binding hormone secreted by adipose tissue, is the only cytokine who is negatively correlated with lipid level in blood serum. Adiponectin plays an important role in regulating lipid metabolism and glycometabolism, ameliorating inflammation. Two adiponectin receptors have been found so far: AipoR1 and AdipoR2. AdipoR2 mostly expressis in liver. So it is supposed that adiponectin may participate in the formation and development of NASH. Thiazolidinediones (rosigliazone) is the specific agonist of peroxisome proliferator-activated receptor gamma (PPARγ). Our previous research showed that rosigliazone could inhibite the hepatic steatosis, inflammation and fibrosis/cirrhosis of mice induced with methionine-choline deficient (MCD) diet. But what is the role of adiponectin in the model is not clear. In this study was to investigate effect of adiponectin in MCD feeding mice and rosigliazone treated mice, which may provide a new approach of NASH therapy through gene modulation.
Objective: In this study, experimental nonalcoholic steatohepatitis models were established by feeding male C57BL6/J mice with methionine-choline deficient (MCD) diet. The treatment group mice were administrated with MCD diet combined with rosigliazone. The expression of adiponectin, tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) were detected to clarify role and mechanism of PPARγagonist, rosigliazone in NASH. This study will provide an theoretical basis for target gene theropy on NASH.
Methods: Experimental models of NASH were established by feeding mice with MCD diet. Thirty healthy male C57BL6/J mice were fed with methionine-choline supplemented diet for one week, and then were randomly divided into three groups: control group, were fed with methionine-choline supplemented diet, model group (MCD group), were given MCD diet, treatment group (MCD+R group), used MCD diet combined with rosiglitazone (50mg/kg/d) for 3 weeks. Serum alanine aminotransferase (ALT) and triglyceride (TG) were tested by enzymic method with automatic biochemistry analyzer. The grade of hepatic steatosis, inflammation and fibrosi were observed by Hematoxylin and eosin, and Masson trichromatism stained paraffin-embedded liver tissues isolated from mice. Adiponectin expression in the liver was examined by immunohistochemistry. The expression of TNF-αand IL-6 mRNA and protein were analyzed by RT-PCR. and western blot, respectively.
Results:
1 The common behavior of mice: control mice were active, their hair was bright and weight increased gradually. Body weight of mice in model group and rosiglitazone group decreased remarkably. Body weight and liver weight in control group, model group and treatment group were respectively :26.00±1.00 g 14.14±0.68 g, 14.31±0.81 g,0.89±0.12 g 0.53±0.10 g 0.56±0.10 g. Body weight and liver weight in model group and treatment group were notedly decreased compared with control group (both P value <0.05). The liver indexs of control group, model group and treatment group was respectively 0.034±0.005, 0.038±0.006, 0.039±0.008, there was no difference among the three groups (P >0.05).
2 The test of biochemical markers of serum: ALT level was markedly elevated in MCD diet mice compared with control mice (50.78±10.03 U/L vs. 161.56±28.23 U/L,P<0.01), and a significant reduction was noticed after rosiglitazone treatment (161.56±28.23 U/L vs. 83.56±12.27 U/L, P<0.01). So does the three groups’serum TG content, control: 0.54±0.29 mmol/L, models: 0.97±0.81 mmol/L, treated group: 0.76±0.13 mmol/L, P <0.05.
3 Liver histopathology : macroscopic observation: Livers in control group were in reddish-brown, with bright gloss. Livers in model group were much smaller than in control group. The whole livers were in light yellow and adhered with other tissues. The cut surface looked reddish-brown without gloss. Livers in treat group were little smaller than in control group and looked reddish-brown with gloss. light microscope observation: Histology of the livers were normal in control mice. Hepatic plates looked well. However, severe steatohepatitis was developed in mice fed with MCD diet, the hepatocytes presented severe macrovesicular steatosis and lacated mainly in zone 3 near the central veins. Spotty necrosis and confluent necrosis can be seen in hepatic lobulars, where mixed inflammatory infiltration with lymphocytes and polymorphonuclear may take place. There were not obvious hepatic fibrosis in perisinusoidal and portal areas (F2-3G2-3S0-1). The degree of hepatic steatosis and inflammation is dramatically ameliorated by administrated rosiglitazone in MCD fed mice (F1G1-2S0-1).
4 The expression of TNF-αmRNA was dramatically improved in MCD fed animals compared with the control group (0.82±0.17 vs. 1.26±0.33,P <0.05), whereas which was dramatically decreased in rosiglitazone treated animals (1.26±0.33 vs. 0.94±0.14,P<0.05).
5 The expression of TNF-αprotein was increased in MCD mice compared with control group (0.47±0.06 vs. 1.21±0.05,P<0.05), whereas which was dramatically decreased in rosiglitazone treated mice (1.21±0.05 vs. 0.83±0.03,P<0.05).
6 The expression of IL-6 mRNA was up-regulated in MCD group (0.34±0.01 vs. 0.89±0.01 , P<0.01) and could be down-regulated by rosiglitazone (0.89±0.01 vs. 0.57±0.03,P<0.05).
7 The expression of IL-6 protein was up-regulated in model animals (0.46±0.01 vs. 0.91±0.01 , P<0.01) and could be down-regulated by rosiglitazone in treatment group (0.91±0.01 vs. 0.79±0.02,P<0.05).
8 Hepatic adiponectin expression: Positive expression was presented around central veins and in endochylema of perisinusoidal cells in the livers of control group. But in model group positive expressions are dramatically enhanced and mainly express in the endochylema of steatosis hepatocytes, kupffer cells in inflammatory infiltration areas and perisinusoidal cells. (1.46±0.27 vs. 15.24±0.36,P<0.01). Compared with model group, the expression of adiponectin was decreased by using rosiglitazone. (15.24±0.36 vs. 9.46±0.74,P< 0.05).
Conclusion:
1 The expressions of adiponectin is dramatically enhanced in livers of the MCD fed mice, which was consistent with expressions of TNF-αand IL-6, which indicates that adiponectin through compensated up-regulation might prevent expression of inflammatory factors and inhibit NASH progression.
2 Rosiglitazone could up-regulate PPARγexpression, further up-regulate adiponectin expression, down-regulate expression of TNF-αand IL-6. Thus, rosiglitazone can inhibit hepatic steatosis, inflammation and fibrosis induced by MCD diet by modulating PPARγ, adiponectin and related inflammatory factor expression.
引文
1 Neuschwander Tetri BA,Caldwell SH.Nonalcoholic steatohepatitis:summary of an AASLD Single Topic Conference [J]. Hepatology, 2003, 37(5):1202- 1219
2 Day CP, James OFW. Steatohepatitis: a tale of two hits [J]? Gastroenterology. 1998, 114:842-845
3 Yamauchi T, Kamon J. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects [J]. Nature, 2003, 423: 762-769
4 Neuschwander Tetri BA, Brunt EM, Wehmeier KR, et al . Improved nonalcoholic steatohepatitis after 48 weeks of treatment with the PPAR- gamma ligand rosiglitazone [J]. Hepatology. 2003, 10: 1008- 1017
5 Zhang BH, Weltman M, Farrell GC. Does steatohepatitis impair liver regeneration? A study in a dietary model of non-alcoholic steatohepatitis in rats [J ]. Gastroenterol Hepatol, 1999, 14 (2): 133- 137
6 Diehl AM, Goodman Z, Ishak KG. Alcohol-like liver disease in nonalcoholics. A clinical and histologic comparison with alcohol-induced liver injury [J]. Gastroenterology, 1998, 95: 1056-1062
7王泰龄,刘霞,周元平等.慢性肝炎炎症活动度及纤维化程度计分方案[J].中华肝脏杂志, 1998, 6: 195- 197
8 Clark JM, Brancati FL, Diehl AM. Nonalcoholic fatty liver disease [J]. Gastroenterology, 2002, 122(6): 1649- 1657
9 Marchesini G, Brizi M,Bianchi G,et a1. Nonalcoholic fatter liver disease: a feature of the metabolic syndrome[J]. Diabetes. 2001, 50(8): 1844- 1850
10 Yodamurakami M, Taniguchi M, Takahashi K, et al. Change in expression of CBP28P adiponectin in carbon tetrachloride-administrated mouse liver [J]. Biochem Biophys Res Commun, 2001, 285(2): 372-377
11刘利梅,赵亚莉,李丽,等.脂联素的信号转导通路[J].生理科学进展, 2005, 36(2): 130-132
12 Yamauchi T, Kamon J,Waki H,et a1.The fat- derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity[J].Nat Med,2001,7(8):941- 946
13 Tilg H, Diehl AM. Cytokines in alcoholic and nonalcoholic steatohepatitis[J]. N Engl J Med, 2000, 343: 1467- 1476
14 Crespo J, Cayon A, Fernandez-Gil P, et al. Gene expression of tumor necrosis factor alpha and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients[J]. Hepatology. 2001, 34: 1158- 1163
15 Stephens JM, Lee J, Pilch PF. Tumor necrosis factor-alpha induced insulin resistence in 3T3-L1 adipocytes is accompanied by a loss of insulin receptor subtrate-1 and GLUT4 expression without a loss of insulin receptor-mediated signal Transduction [J]. J Biol chem, 1997, 272(2): 971~976
Stephens JM, Pekala PH. Transcriptional repression of the C/EBP-alpha and GLUT4 Genes in 3T3-L1 adipocytes by tumor necrosis factor-alpha. Regulation is coordinate and independent of protein synthesis [J]. J Biol Chem, 1992, 267(19): 13580- 13584
17 Olefsky JM, Nolan JJ. Insulin resistance and non-insulin-dependent diabetes mellitus: cellular and molecular mechanisms [J]. Am J Clin Nutr 1995,61: 980S~986S
18 Kim HJ, Higashimori T, Park SY,et al. Differential effects of interleukin-6 and -10 on skeletal muscle and liver insulin action in vivo[J]. Diabetes. 2004; 53: 1060-1067
19 Kugelmas M, Hill DB, Vivian B, et al. Cytokines and NASH: a pilot studyof the effects of lifestyle modification and vitamin E[J]. Hepatology. 2003; 38: 413- 419
20 Lutchman G, Promrat K, Kleiner DE, et al. Changes in serum adipokine levels during pioglitazone treatment for nonalcoholic steatohepatitis: relationship to histological improvement [J]. Clin Gastroenterol Hepatol. 2006; 4: 1048- 1052
21 Pagano C, Soardo G, Esposito W, et al. Plasma adiponectin is decreased in nonalcoholic fatty liver disease [J]. Eur Endocrinol 2005; 152: 113-118
22 Vuppalanchi R, Marri S, Kolwankar D, et al. Is adiponectin involved in the pathogenesis of nonalcoholic steatohepatitis? A preliminary human study [J]. Clin Gastroenterol 2005; 39: 237-242
23 Hui JM, Hodge A, Farrell GC, et al. Beyond insulin resistance in NASH: TNFalpha or adiponectin [J]? Hepatology 2004; 40: 46-54
24 Maeda N, Shimomura I, Kishida K, et al. Diet-induced insulin resistance in mice lacking adiponectin/ACRP30 [J]. Nat Med. 2002, 8: 731- 737
25魏永杰,张华.细胞因子在非酒精性脂肪性肝炎发病机制中的作用[J].国际消化病杂志. 2006. 26(1): 14-16
26 Kahn BB, Alquier T, Carling D, et al. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism [J]. Cell Metab 2005; 1: 15- 25
27 Kola B, Boscaro M, Rutter GA, et al. Expanding role of AMPK in endocrinology. Trends Endocrinol Metab 2006; 17: 05- 15
28 Viollet B, Foretz M, Guigas B, et al. Activation of AMP-activated protein kinase in the liver: a new strategy for the management of metabolic hepatic disorders [J]. Physiol 2006; 574: 41- 53
29 Fasshauer M, Kralisch S, Klier M, et al. Adiponectin gene expression and secretion is inhibited by interleukin-6 in 3T3-L1 adipocytes[J]. Biochem Biophys Res Commun. 2003, 301: 10 45–50
1 Yoda murakami M, Taniguchi M, Takahashi K, et al. Change in expression of CBP28P adiponectin in carbon tetrachloride-administrated mouse liver[J]. Biochem Biophys Res Commun, 2001, 285(2): 372-377
2 Scherer PE, Williams S, Fogliano M, et al. A novel serum protein similar to C1q, produced exclusively in adipocytes [J]. Biol Chem, 1995, 270(45): 26746-26749
3 Hu E, Liang P, Spiegelman BM. AdipoQ is a novel adipose-specific gene dysregulated in obesity[J]. Biol Chem, 1996, 271(18): 10697-10703
4 Maeda K, Okubo K, Shimomura I, et al. cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (Adipose most abundant gene transcript 1 ) [J]. Biochem Biophys Res Commun, 1996, 221(2): 286-289
5 Wang Y, Xu A, Knight C, et al. Hydroxylation and glycosylation of the four conserved lysine residues in the collagenous domain of adiponectin: potential role in the modulation of its insulin-sensitizing activity [J]. Biol Chem, 2002, 277(22): 19521-19529
6 Yamauchi T, Kamon J. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects [J]. Nature, 2003, 423: 762-769
7 Yalniz M, Bahcecioglu IH, Ataseven H, et al. Serum adipokine and ghrelin levels in nonalcoholic steatohepatitis. Mediators Inflamm 2006; 2006: 34295
8刘利梅,赵亚莉,李丽,等.脂联素的信号转导通路[J].生理科学进展, 2005, 36(2): 130-132
9 Philip M, Alyssa C, Ashley N, et al. p38 Mitogen-activated Protein Kinase Activates Peroxisome Proliferator-activarted Receptor a [J]. Biol Chem, 2001, 276: 44495-44501
10 You M, Matsumoto M. The role of AMP-activated protein kinase in the action of ethanol in the liver [J]. Gastroenterology, 2004, 127: 1798-1808
11 Xu A, Wang Y, Hussila K, et al. The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice [J]. Clin Invest, 2003, 112: 91- 100
12洪洁,顾卫琼,张翼飞,等.胰岛素抵抗综合征患者血清脂联素和胰岛素敏感性的相关性研究[J].中华内分泌代谢杂志, 2003, 19: 173-176
13卢慧玲,王宏伟,林汉华,等.血浆脂联素与2型糖尿病胰岛素抵抗关系的研究[J].临床内科杂志, 2003, 20: 594-596
14 Kubota N, Terauchi Y, yamauchi T, et al. Disruption of adiponectin causes insulin resistance and neointimal formation [J]. Biol chem, 2002, 227: 25863-25866
15魏永杰,张华.细胞因子在非酒精性脂肪性肝炎发病机制中的作用[J].国际消化病杂志. 2006. 26(1): 14-16
16 Soares AF, Guichardant M, Cozzone D, et al. Effects of oxidative stress on adiponectin secretion and lactate production in 3T3-L1 adipocytes [J]. Free Radic Biol Med, 2005, 38: 882-889
17薛建波,杨丽.脂联素与肝纤维化[J],医学综述, 2007, 13(1): 116-118
18 Arita Y, Kihara S, Ouchi N, et al. Adipocyte-derived plasma protein adiponectin acts as a platelet-derived growth factor-BB-binding protein and regulates growth factor induced common post receptor signal in vascular smooth muscle cell [J]. Circulation, 2002, 105(24): 2893-2898
19 Ding X, Saxena NK, Lin S, et al. The roles of leptin and adiponectin: a novel paradigm in adipocytokine regulation of liver fibrosis and stellate cell biology [J]. AmJ Pathol, 2005, 166(6): 1655-1669
20 Thakur V, Pritchard MT, McMullen MR, et al. Adiponectin normalizes LPS-stimulated TNF-αproduction by rat Kupffer cells after chronic ethanol feeding [J]. Am J Physiol Gastrointest Liver Physiol, 2006, 290(5): G998-1007
21 Musso G, Gambino R, Biroli G, et al. Hypoadiponectinemia predicts the severity of hepatic fibrosis and pancreatic Beta-cell dysfunction in nondiabetic nonobese patients with nonalcoholic steatohepatitis [J]. Am J Gastroenterol 2005, 100(11): 2438-2446
22 Vuppalanchi R, Marri S, Kolwankar D, et al. Is adiponectin involved in the pathogenesis of nonalcoholic steatohepatitis? A preliminary human study [J]. J Clin Gastroenterol 2005: 39(3): 237-242
2 Day CP, James OFW. Steatohepatitis: a tale of two hits [J]? Gastroenterology. 1998, 114:842-845
3 Yamauchi T, Kamon J. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects [J]. Nature, 2003, 423: 762-769
4 Neuschwander Tetri BA, Brunt EM, Wehmeier KR, et al . Improved nonalcoholic steatohepatitis after 48 weeks of treatment with the PPAR- gamma ligand rosiglitazone [J]. Hepatology. 2003, 10: 1008- 1017
5 Zhang BH, Weltman M, Farrell GC. Does steatohepatitis impair liver regeneration? A study in a dietary model of non-alcoholic steatohepatitis in rats [J ]. Gastroenterol Hepatol, 1999, 14 (2): 133- 137
6 Diehl AM, Goodman Z, Ishak KG. Alcohol-like liver disease in nonalcoholics. A clinical and histologic comparison with alcohol-induced liver injury [J]. Gastroenterology, 1998, 95: 1056-1062
7王泰龄,刘霞,周元平等.慢性肝炎炎症活动度及纤维化程度计分方案[J].中华肝脏杂志, 1998, 6: 195- 197
8 Clark JM, Brancati FL, Diehl AM. Nonalcoholic fatty liver disease [J]. Gastroenterology, 2002, 122(6): 1649- 1657
9 Marchesini G, Brizi M,Bianchi G,et a1. Nonalcoholic fatter liver disease: a feature of the metabolic syndrome[J]. Diabetes. 2001, 50(8): 1844- 1850
10 Yodamurakami M, Taniguchi M, Takahashi K, et al. Change in expression of CBP28P adiponectin in carbon tetrachloride-administrated mouse liver [J]. Biochem Biophys Res Commun, 2001, 285(2): 372-377
11刘利梅,赵亚莉,李丽,等.脂联素的信号转导通路[J].生理科学进展, 2005, 36(2): 130-132
12 Yamauchi T, Kamon J,Waki H,et a1.The fat- derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity[J].Nat Med,2001,7(8):941- 946
13 Tilg H, Diehl AM. Cytokines in alcoholic and nonalcoholic steatohepatitis[J]. N Engl J Med, 2000, 343: 1467- 1476
14 Crespo J, Cayon A, Fernandez-Gil P, et al. Gene expression of tumor necrosis factor alpha and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients[J]. Hepatology. 2001, 34: 1158- 1163
15 Stephens JM, Lee J, Pilch PF. Tumor necrosis factor-alpha induced insulin resistence in 3T3-L1 adipocytes is accompanied by a loss of insulin receptor subtrate-1 and GLUT4 expression without a loss of insulin receptor-mediated signal Transduction [J]. J Biol chem, 1997, 272(2): 971~976
Stephens JM, Pekala PH. Transcriptional repression of the C/EBP-alpha and GLUT4 Genes in 3T3-L1 adipocytes by tumor necrosis factor-alpha. Regulation is coordinate and independent of protein synthesis [J]. J Biol Chem, 1992, 267(19): 13580- 13584
17 Olefsky JM, Nolan JJ. Insulin resistance and non-insulin-dependent diabetes mellitus: cellular and molecular mechanisms [J]. Am J Clin Nutr 1995,61: 980S~986S
18 Kim HJ, Higashimori T, Park SY,et al. Differential effects of interleukin-6 and -10 on skeletal muscle and liver insulin action in vivo[J]. Diabetes. 2004; 53: 1060-1067
19 Kugelmas M, Hill DB, Vivian B, et al. Cytokines and NASH: a pilot studyof the effects of lifestyle modification and vitamin E[J]. Hepatology. 2003; 38: 413- 419
20 Lutchman G, Promrat K, Kleiner DE, et al. Changes in serum adipokine levels during pioglitazone treatment for nonalcoholic steatohepatitis: relationship to histological improvement [J]. Clin Gastroenterol Hepatol. 2006; 4: 1048- 1052
21 Pagano C, Soardo G, Esposito W, et al. Plasma adiponectin is decreased in nonalcoholic fatty liver disease [J]. Eur Endocrinol 2005; 152: 113-118
22 Vuppalanchi R, Marri S, Kolwankar D, et al. Is adiponectin involved in the pathogenesis of nonalcoholic steatohepatitis? A preliminary human study [J]. Clin Gastroenterol 2005; 39: 237-242
23 Hui JM, Hodge A, Farrell GC, et al. Beyond insulin resistance in NASH: TNFalpha or adiponectin [J]? Hepatology 2004; 40: 46-54
24 Maeda N, Shimomura I, Kishida K, et al. Diet-induced insulin resistance in mice lacking adiponectin/ACRP30 [J]. Nat Med. 2002, 8: 731- 737
25魏永杰,张华.细胞因子在非酒精性脂肪性肝炎发病机制中的作用[J].国际消化病杂志. 2006. 26(1): 14-16
26 Kahn BB, Alquier T, Carling D, et al. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism [J]. Cell Metab 2005; 1: 15- 25
27 Kola B, Boscaro M, Rutter GA, et al. Expanding role of AMPK in endocrinology. Trends Endocrinol Metab 2006; 17: 05- 15
28 Viollet B, Foretz M, Guigas B, et al. Activation of AMP-activated protein kinase in the liver: a new strategy for the management of metabolic hepatic disorders [J]. Physiol 2006; 574: 41- 53
29 Fasshauer M, Kralisch S, Klier M, et al. Adiponectin gene expression and secretion is inhibited by interleukin-6 in 3T3-L1 adipocytes[J]. Biochem Biophys Res Commun. 2003, 301: 10 45–50
1 Yoda murakami M, Taniguchi M, Takahashi K, et al. Change in expression of CBP28P adiponectin in carbon tetrachloride-administrated mouse liver[J]. Biochem Biophys Res Commun, 2001, 285(2): 372-377
2 Scherer PE, Williams S, Fogliano M, et al. A novel serum protein similar to C1q, produced exclusively in adipocytes [J]. Biol Chem, 1995, 270(45): 26746-26749
3 Hu E, Liang P, Spiegelman BM. AdipoQ is a novel adipose-specific gene dysregulated in obesity[J]. Biol Chem, 1996, 271(18): 10697-10703
4 Maeda K, Okubo K, Shimomura I, et al. cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (Adipose most abundant gene transcript 1 ) [J]. Biochem Biophys Res Commun, 1996, 221(2): 286-289
5 Wang Y, Xu A, Knight C, et al. Hydroxylation and glycosylation of the four conserved lysine residues in the collagenous domain of adiponectin: potential role in the modulation of its insulin-sensitizing activity [J]. Biol Chem, 2002, 277(22): 19521-19529
6 Yamauchi T, Kamon J. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects [J]. Nature, 2003, 423: 762-769
7 Yalniz M, Bahcecioglu IH, Ataseven H, et al. Serum adipokine and ghrelin levels in nonalcoholic steatohepatitis. Mediators Inflamm 2006; 2006: 34295
8刘利梅,赵亚莉,李丽,等.脂联素的信号转导通路[J].生理科学进展, 2005, 36(2): 130-132
9 Philip M, Alyssa C, Ashley N, et al. p38 Mitogen-activated Protein Kinase Activates Peroxisome Proliferator-activarted Receptor a [J]. Biol Chem, 2001, 276: 44495-44501
10 You M, Matsumoto M. The role of AMP-activated protein kinase in the action of ethanol in the liver [J]. Gastroenterology, 2004, 127: 1798-1808
11 Xu A, Wang Y, Hussila K, et al. The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice [J]. Clin Invest, 2003, 112: 91- 100
12洪洁,顾卫琼,张翼飞,等.胰岛素抵抗综合征患者血清脂联素和胰岛素敏感性的相关性研究[J].中华内分泌代谢杂志, 2003, 19: 173-176
13卢慧玲,王宏伟,林汉华,等.血浆脂联素与2型糖尿病胰岛素抵抗关系的研究[J].临床内科杂志, 2003, 20: 594-596
14 Kubota N, Terauchi Y, yamauchi T, et al. Disruption of adiponectin causes insulin resistance and neointimal formation [J]. Biol chem, 2002, 227: 25863-25866
15魏永杰,张华.细胞因子在非酒精性脂肪性肝炎发病机制中的作用[J].国际消化病杂志. 2006. 26(1): 14-16
16 Soares AF, Guichardant M, Cozzone D, et al. Effects of oxidative stress on adiponectin secretion and lactate production in 3T3-L1 adipocytes [J]. Free Radic Biol Med, 2005, 38: 882-889
17薛建波,杨丽.脂联素与肝纤维化[J],医学综述, 2007, 13(1): 116-118
18 Arita Y, Kihara S, Ouchi N, et al. Adipocyte-derived plasma protein adiponectin acts as a platelet-derived growth factor-BB-binding protein and regulates growth factor induced common post receptor signal in vascular smooth muscle cell [J]. Circulation, 2002, 105(24): 2893-2898
19 Ding X, Saxena NK, Lin S, et al. The roles of leptin and adiponectin: a novel paradigm in adipocytokine regulation of liver fibrosis and stellate cell biology [J]. AmJ Pathol, 2005, 166(6): 1655-1669
20 Thakur V, Pritchard MT, McMullen MR, et al. Adiponectin normalizes LPS-stimulated TNF-αproduction by rat Kupffer cells after chronic ethanol feeding [J]. Am J Physiol Gastrointest Liver Physiol, 2006, 290(5): G998-1007
21 Musso G, Gambino R, Biroli G, et al. Hypoadiponectinemia predicts the severity of hepatic fibrosis and pancreatic Beta-cell dysfunction in nondiabetic nonobese patients with nonalcoholic steatohepatitis [J]. Am J Gastroenterol 2005, 100(11): 2438-2446
22 Vuppalanchi R, Marri S, Kolwankar D, et al. Is adiponectin involved in the pathogenesis of nonalcoholic steatohepatitis? A preliminary human study [J]. J Clin Gastroenterol 2005: 39(3): 237-242